The objective of the proposed work is to push the boundaries of biological imaging, so that it will be routinely possible to image specific cellular molecules with exceptional spatial resolution (2-3 nm) and high sensitivity (i.e., single molecules can be detected). If we are successful, our technologies will allow any cell biologist to study any biological process, inside living cells, with unprecedented accuracy. Current technology allows detection of single molecules in vitro but not routinely inside living cells, and sub- diffraction far-field optical imaging can currently be performed at 20-30 nm resolution on fixed, but not living cells. In our view, the major challenge to improving single-molecule and super-resolution imaging is the lack of suitable fluorescent probes. We plan to solve this problem in three parts. First, we will design and synthesize new fluorescent probes which are extremely bright and photostable, but can also be delivered into the cytoplasm of living cells and remain well-solubilized. Second, we will develop novel methods for conjugating these fluorophores to specific cellular proteins of interest. Third, we will develop physical methods for enhancing the fluorescence emission of existing fluorophores, while minimizing background from cellular autofluorescence. All these new molecules and techniques will be collectively applied to perform ultra-sensitive, high resolution optical imaging on fixed and living cells. We also have a special interest in neuroscience, and will apply the new optical imaging methods that we develop to study the trafficking and localization of neurotransmitter receptors that play a central role in learning and memory formation. Public Health Relevance: If successful, our research will produce imaging technologies that will benefit the entire community of cell biologists, by allowing single-molecule and super-resolution optical imaging of biological processes in living cells with unprecedented detail. If our proposed experiments on neurotransmitter receptor imaging are successful, we will gain fundamental insight into the molecular mechanisms of learning and memory and neuronal development, which will in turn shed light on neurologic and psychiatric disorders including stroke, epilepsy, brain injury, addiction, schizophrenia, autism, and chronic pain.